Ku autoantigen is a DNA binding protein consisting of 7O-kDa (Ku70) and 86-kDa (Ku80) subunits. Recent data have implicated Ku80 in the repair of DNA double strand breaks, and Ku70 in the suppression of heat-induced hsp70 expression. The long term goal of this project is to understand the molecular bases of the cellular response to stress. Work in the next granting period will focus on the mechanistic and functional aspects of Ku protein, in modulating cellular response to heat shock and to ioizing radiation. There are three specific aims: Specific Aim I focuses on the molecular mechanism(s) by which Ku suppresses heat-induction of hsp70. We will test a hypothesis inferred from our preliminary studies that Ku regulates the heat shock response by modulating the binding of transcription factors to their respective regulatory elements in the promoter region of the hsp70 gene. By in vivo genomic footprinting, the effects of Ku on the pattern of protein binding to the hsp70 promoter, especially its effects on the binding of the heat shock transcription activator HSF1 and other transcription factors to their respective binding sites will be determined. The possibility that Ku binds to a specific element in the regulatory region of the hsp70 gene will be tested. In addition to in vivo genomic footprinting, mutations will be introduced into the promoter of hsp70, linked to a reporter gene construct, to test for the presence of a cis-element(s), which when mutated abolishes the Ku-mediated suppression of heat-induction of reporter gene expression. In Specific Aim II we will study the structural and functional domains of Ku that are involved in the modulation of hsp70 gene expression. Rodent cell lines stably and constitutively expressing various mutant Ku genes will be established. The heat shock response of these cells and the biochemical properties of wild type and mutant Ku subunits will be examined. In Specific Aim III we propose to establish homozygous rodent cell lines in which both alleles of Ku70 or Ku80 are inactivated through gene-targeting mutagenesis. These "double knockout" Ku7O-/- and Ku8- /- cell lines will enable us to study various physiological roles of the individual subunits of Ku and to dissect the functional domains involved in the cellular response to heat shock and ionizing radiation. Because of the biological importance and the multiple cellular roles of Ku, the significance of constructing Ku70-/- and Ku80-/- lines extends beyond the immediate goal of elucidating the role of Ku in hsp70 gene regulation, and in the sensitivity of cells to x-rays. These cell lines and their derivatives expressing various mutant Ku subunits can be used directly in the analysis of any functional aspect of Ku. The choice of embryonic stem cells for this work further assures that such studies can be readily extended to animal models, through the construction of knockout mice strains.